Immunoassay and immunometric assay of free ligand concentrations in biological fluids

ABSTRACT

A method of measuring the concentration of a free ligand in a biological fluid containing the free ligand and ligand bound to endogenous binding agent, by the steps of 
     (a) mixing a sample of the fluid with an analogue of the ligand, a specific binder with which the free ligand and the ligand analogue bind, and an exogenous binding agent which binds the ligand analogue but not the ligand, either the ligand analogue or the specific binder being labelled, 
     (b) incubating the resulting mixture, 
     (c) determining either the amount of the labelled analogue bound or the amount of labelled specific binder bound, or not bound, to the ligand analogue, and 
     (d) correlating the determined amount to the amount of free ligand present in the sample. 
     The method is useful to measure concentration of free thyroid hormones and other hormones in body fluids, employing antibodies specific to the ligand analogue as the exogenous binding agents.

TECHNICAL FIELD

The present invention relates to a method for measuring theconcentration of free ligand in a biological fluid containing freeligand and ligand bound to endogenous binding agent.

BACKGROUND ART

Immunoassay techniques have been developed in recent vears to measureconcentrations of free hormones and other ligands in sera and otherbiological fluids which contain free ligand in equilibrium with ligandbound to endogenous binding agents such as binding proteins. They arebased on the principle that if a specific binder for the ligand, usuallyan antibody, is brought into contact with the sample to be tested theextent of occupancy of the binding sites on the specific binder by theligand is a measure of the concentration of free ligand, provided thatthe amount of specific binder is sufficiently low that the equilibriumbetween free and endogenously bound ligand is not significantlyaffected. By measuring the extent of occupancy for the unknown sampleand calibrating such a measurement using standard samples containingknown free ligand concentrations it is possible to determine the freeligand concentration in the unknown sample.

Initially, the extent of occupancy of binding sites was measured byremoving the specific binder containing bound ligand from the sample anddeterming the proportion of unoccupied sites by back-titration using anappropriately labelled material (e.g. radioactively labelled material)which binds at the unoccupied sites. The process was thus effectively atwo-step process.

Subsequently it has been proposed to carry out the `back-titration`without removing the specific binder from the sample, thus convertingthe two-step process into a one-step process. This can be done either byusing as the labelled material a labelled analogue of the ligand or byusing as the labelled material a specific binding agent.

Thus, it has been proposed in published European Patent Application No.0,026,103 which is equivalent to U.S. Pat. No. 4,366,143 of Midgley etal to measure the concentration of free ligand in such a biologicalfluid by a radioimmunoassay technique comprising (a) admixing a sampleof the fluid with a labelled derivative of the ligand and with aspecific binder for the ligand, (b) effecting reaction between the freeligand, the labelled derivative and the specific binder, (c) ifnecessary, separating that portion of the ligand and labelled derivativethat has become bound to the specific binder from that portion not sobound, (d) measuring the amount of the labelled derivative that is, oris not, bound to the specific binder, and (e) using that measurement todetermine the concentration of free ligand in the biological fluid.According to the process disclosed there, the labelled derivative of theligand is chosen to bind strongly to the added specific binder but tobind not at all, or much more weakly than does the ligand, to theendogenous binding agent.

In an alternative procedure a method of determining the free ligandconcentration involves an immunoradiometric assay comprising admixing asample of the fluid with a labelled specific binder and an unlabelledanalogue of the ligand, incubating the resulting mixture to permit thefree ligand and the unlabelled analogue to compete for the labelledspecific binder, determining the amount of labelled specific binderbound either to the ligand or to the unlabelled ligand analogue, andcorrelating the amount of bound labelled specific binder to the amountof free ligand present in the sample.

However when practical assay kits embodying the principles of EPA No.0,026,103 have been employed to assay free thyroid hormone in samplestaken from patients suffering, for example, from certain non-thyroidalillnesses or having serum protein abnormalities unrelated to freethyroid hormone concentration, the assay results appear to show ananomalous free thyroid hormone concentration, contrary to the correctposition. It has also been found that the concentration of antibody(acting as specific binder) in those kits can be up to 100 times greaterthan would have been expected on the simplified theoretical explanationof this technique hitherto proposed.

Further investigation into the operation of those kits has revealedthat, far from the ligand analogue being totally unbound to endogenousbinding agents or being bound to only a small extent, it is bound to avery substantial extent, at least 90% and probably as much as 99%, notonly to the albumin present in the sample but also to the other bindingproteins TBG and TBPA.

It is therefore an object of the present invention to devise analternative and improved technique for assaying free ligandconcentrations which is not subject to the disadvantages inherent in theprevious technique.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram illustrating the present invention in terms ofcompeting equilibrium reactions, and

FIG. 2 is a graph showing the extent of binding of the analogue to thebinding reagent.

DISCLOSURE OF INVENTION

It has now been found that, for the immunoassay technique using labelledligand analogue, when any fraction of the ligand analogue becomes boundto other binding agents in the sample as well as to the specific binderthe fraction, b, of the ligand analogue bound to the specific binder isrepresented by the following equation (provided that the concentrationof specific binder is sufficiently low that the equilibrium between freeand bound ligand in the sample is not significantly disturbed): ##EQU1##where, K_(H) is the equilibrium constant for the ligand/specific binderreaction,

K_(An) is the equilibrium constant for the analogue/specific binderreaction,

[fH] is the free ligand concentration,

[An] is the analogue concentration,

[Ab] is the specific binder concentration,

    ΣK.sub.p [p] is K.sub.p1 [P.sub.1 ]+K.sub.p.sbsb.2 [P.sub.2 ] . . . +K.sub.p.sbsb.n [P.sub.n ],

[P₁ ], [P₂ ] . . . [P_(n) ] are the concentrations of the various(endogenous or added) binding agents other than Ab in the sample, and

K_(p).sbsb.1, K_(p).sbsb.2 . . . K_(p).sbsb.n are the correspondingequilibrium constants for the reactions between the ligand analogue andthe various (endogenous or added) binding agents.

A similar equation, in which the term 1+ΣK_(p) [P]--hereafter referredto as S--also appears, can be formulated for the immunometric assaytechnique in which labelled binding agent is used.

On the basis of this theoretical equation it is possible to design animproved assay technique.

Firstly, in situations where the ligand analogue binds with endogenousbinding agent and the extent of binding varies from sample to sample ofthe biological fluid because of variations in the concentration ofendogenous binding agent and/or the equilibrium constant for theanalogue/endogenous binding agent reaction, it is possible to reduce thesignificance of those variations by adding a further binding agent (X)having a concentration [P_(x) ] and an equilibrium constant K_(p).sbsb.xfor reaction with the analogue such that K_(p).sbsb.x [P_(x) ]contributes significantly to the term S and the contribution to thatterm from the products K_(p).sbsb.1 [p₁ ] . . . K_(p).sbsb.n [p_(n) ]for the endogenous binding agent(s) is proportionately reduced.

Secondly, even in situations where the ligand analogue does not bind atall or to any significant extent with endogenous binding agent presentin the sample or where the concentrations and equilibrium constants forendogenous binding agent do not vary significantly from sample tosample, the addition of a further binding agent for the ligand analogueenables the term S to be increased. The constraints imposed by theequation then allow [Ab] to be increased without sacrificing theaccuracy or responsiveness of the technique, which means that the assaycan be completed more quickly or that it is possible to use a specificbinder of lower specific activity.

According to the invention therefore there is provided an improvedmethod of measuring the concentration of a free ligand in a biologicalfluid containing the free ligand and ligand bound to endogenous bindingagent, comprising

(a) mixing a sample of the fluid with an analogue of the ligand, aspecific binder with which the free ligand and the ligand analogue bind,and an exogenous binding agent which binds the ligand analogue but notthe ligand, either the ligand analogue or the specific binder beinglabelled,

(b) incubating the resulting mixture so that the ligand and ligandanalogue compete for the specific binder,

(c) determining either the amount of the labelled analogue bound to thespecific binder or the exogenous binding agent or the amount of labelledspecific binder bound, or not bound, to the ligand analogue, and

(d) correlating the determined amount to the amount of free ligandpresent in the sample.

The exogenous binding agent functions in the method of the presentinvention as a buffering system for the ligand analogue, having theeffect of reducing or eliminating irrelevant fluctuations in thecomposition of the fluids being tested. Essentially the ligand analogueis the subject of competition between two binding agents, namely thespecific binder--hereafter referred to as A--which is also a binder forthe ligand, and the exogenous binding agent--hereinafter referred to asX--which is not a binder for the ligand.

Where the contribution to S from the endogenous binding agents is zeroor is substantially constant for all samples the exogenous binding agentX and its concentration are advantageously chosen so that K_(p).sbsb.x[P_(x) ] and S are both at least 10, S preferably being 50-500. When thecontribution to S from the endogenous binding agents is liable to varysignificantly from sample to sample the exogenous binding agent X andits concentration are advantageously chosen so that K_(p).sbsb.x [P_(x)] is comparable with or larger than the expected variation in S andpreferably constitutes at least half of S, for example two thirds tonine tenths of S.

It will be appreciated however that the ranges of optimum utility forthe product K_(p).sbsb.x [p_(x) ] as a fraction of S will vary from caseto case depending on the clinical acceptability of inaccuracies in themeasurement of free ligand and the extent to which the contribution to Sfrom the endogenous binding agents alone is likely to vary.

A practical upper limit on the amount of exogenous binding agent X mayoften be imposed by the fact that increases in the amount of theexogenous binding agent X will in general be accompanied by increases inthe amount of the specific binder A and that too great an increase inthe amount of the specific binder A will lead to a significantdisturbance of the equilibrium between free and bound ligand in thebiological fluid.

Expressed in terms of competing equilibrium reactions, the method of thepresent invention can be depicted as shown in FIG. 1 of the accompanyingdrawings. An equilibrium is set up between free ligand, endogenouslybound ligand, ligand bound to specific binder A, free ligand analogue,ligand analogue bound to specific binder, ligand analogue bound toexogenous binding agent X and, in the usual case, endogenously boundligand analogue. Thus the invention differs from the system described inEPA No. 0,026,103 by the provision of a ligand analogue which can beextensively bound to endogenous binding agents and is buffered by thepresence of the additional exogenous binding agent X so that the effectsof fluctuations in the equilibrium reactions with endogenous bindingagents can be proportionately reduced.

The choice of the exogenous binding agent X is dependent upon the natureof the ligand and the ligand analogue because it is essential that itshould bind with the ligand analogue and not with the ligand. It is alsoan essential requirement for this binding agent, as for the specificbinder A, that it must not through its inherent nature or itsconcentration disturb the equilibrium between the free ligand and theendogenously bound ligand, nor must it in turn be influenced by theendogenous binding agent or by drugs or any other ingredients likely tobe present in the fluids being tested. The exogenous binding agent X mayfor example be a physical encapsulation of the analogue. Preferably,however, it will be a reagent, especially an antibody, which is talloredaccording to the chemical differentiation between the ligand andanalogue so as to bind the latter but not the former. Advantageously,the antibody acting as binding agent X does not have a very highaffinity for the analogue (provided that its affinity for the ligand islower by at least about 2 orders of magnitude). Such antibodies may thenbe used at fairly high concentrations to provide the required value forthe product K_(p).sbsb.x [p_(x) ]. Those familiar with immunoassaytechniques will be able to design an appropriate exogenous binding agentX without difficulty.

The method of the present invention is applicable not only toimmunoassay techniques (eg. radioimmunoassay) in which the ligandanalogue is labelled (eg. radioactively) but also to immunometric assaytechniques (e.g. immunoradiometric assays) in which the specific binderis labelled (e.g. radioactively).

The method may be used to measure concentrations of free hormones inbiological fluids, especially free thyroid hormones T₃ and T₄ but alsoother hormones such as cortisol, progesterone, oestradiol andtestosterone. The specific binders A used may be those known to beuseful for this purpose in previous immunoassay techniques or may beformulated according to known principles. The ligand analogues describedin EPA No. 0,026,103 and No. 0,073,865 may be used in the method of thepresent invention, as may other ligand analogues and it will beappreciated that it is no longer necessary to attempt to design a ligandanalogue which will not be bound to endogenous binding agents but merelyone which can be bound to a binding agent X which does not bind theligand itself. The ligand analogues can be labelled in any appropriatemanner, for example as described in EPA No. 0,026,103 when immunoassaytechniques are to be used. Alternatively, the analogues may be used inan unlabelled state together with a labelled specific binder asdescribed in greater detail in International patent application WO83/03306. The other operational conditions appropriate for the method ofthe present invention may be the same as those known or conventional inprevious immunoassay techniques.

The invention and the improvement achievable by its use, are illustratedby the following example.

EXAMPLE

An analogue of thyroxine (T₄) suitable for the immunoassay of free T₄(fT₄) as described in EPA No. 0,026,103 was prepared by chemicalmodification of the amino acid structure of T₄. An antibody (X) againstthis analogue was produced by well known immunological techniques andshown to have a relative affinity for analogue as compared to itsaffinity for T₄ of 10³.

The analogue was radiolabelled with ¹²⁵ I by the well known "exchange"method and shown to have much lower affinity constants than T₄ for thenormal T₄ binding proteins thus satisfying the requirements of EPA No.0,026,103.

A specific antibody against T₄ (A), with an equal affinity for themodified T₄ analogue was coupled to solid particles.

A mixture was prepared of 0.5 ml of a suspension of the solid-phaseantibody reagent (2 nm) and 0.5 ml of the ¹²⁵ I T₄ analogue (2 nM), bothdiluted in 4% BSA, PBS pH 7.4 and a 100 ul aliquot of normal human serumcontaining various concentrations of fT₄ (prepared by well knowntechniques). The extent of binding of the ¹²⁵ I analogue to the specificbinding reagent was correlated with fT₄ concentration as shown in FIG.2(a).

A similar mixture was prepared containing identical concentrations ofspecific antibody and analogue but with 100 ul aliquots of samplescontaining 3 nM oleic acid and varying concentrations of fT₄. (Oleicacid is one of a class of compounds known as non-esterified-fatty acidswhich are known to be increased in serum samples following theadministration of some drugs and during non-thyroidal illness.)

The extent of binding was found to correlate with fT₄ as shown in FIG.2(b). Thus a sample containing, e.g. 20 pM fT₄ and 3 mM oleic acidwould, because of the increase in the extent of analogue binding, beinterpreted as containing 10.6 pM fT₄, a bias of 47%.

The K_(p).sbsb.n [P_(n) ] in the incubation conditions of this assay isestimated to be 70.

According to the method of this invention, the additional binding agentprepared as previously described, was added to identical mixtures ofantibody and antigen at a concentration of 0.2% such that ΣK_(x) [P_(x)]=150. Again the extent of binding of the labelled analogue with thespecific antibody was correlated with the fT₄ concentration before andafter the addition of 3 mM oleic acid as shown in FIG. 2(c and d)respectively.

In this example of the invention a sample containing 20 pg/ml fT₄ and 1mM oleic acid would be interpreted as containing 17 pg/ml fT₄, anegative bias of only 15%. Further increases in the addition of thebinding agent cause additional decreases in the observed bias of themethod and the required concentration of binding agent will depend onthe bias permissible in the estimation of fT₄ for clinical reasons.

We claim:
 1. A method of measuring the concentration of free ligand in abiological fluid containing the ligand both as a free ligand and asligand reversibly bound to an endogenous binding agent, comprising(a)mixing a sample of the biological fluid with (1) an analogue of theligand, (2) a specific binder with which the free ligand and the ligandanalogue reversibly bind, and (3) an exogenous binding agent whichreversibly binds the ligand analogue but not the ligand, either theligand analogue or the specific binder being labelled, (b) incubatingthe resulting mixture so that the ligand and ligand analogue compete forthe specific binder, (c) determining either (1) the amount of thelabelled analogue bound to the specific binder or the exogenous bindingagent; or (2) the amount of labelled specific binder bound, or notbound, to the ligand analogue, and (d) correlating the determined amountto the amount of free ligand present in the biological fluid.
 2. Amethod as claimed in claim 1 wherein the contribution from theendogenous binding agents to S, wherein S is defined as one plus the sumof the products of the concentrations of each of the binding agents inthe sample other than the specific binder and the respective equilibriumconstants for the reactions between those binding agents and the ligandanalogue, is zero or constant for all samples and the exogenous bindingagent and its concentration are selected so that its contribution to Sis at least 10, S being not more than
 500. 3. A method as claimed inclaim 1 wherein the contribution from the endogenous binding agents toS, wherein S is defined as one plus the sum of the products of theconcentrations of each of the binding agents in the sample other thanthe specific binder and the respective equilibrium constants for thereactions between those binding agents and the ligand analogue and Svaries significantly from sample to sample and the exogenous bindingagent and its concentration are chosen so that its contribution to S isat least half the value of S in any sample and is comparable with orgreater than the variation in S from sample to sample.
 4. A method asclaimed in claim 1 wherein the exogenous binding agent is a chemicalreagent which binds the ligand analogue but does not substantially bindthe ligand.
 5. A method as claimed in claim 4 wherein the exogenousbinding agent is an antibody having an affinity for the ligand analogueat least two orders of magnitude higher than its affinity for theligand.
 6. A method as claimed in claim 1 wherein the ligand analogue islabelled.
 7. A method as claimed in claim 1 wherein the specific binderis labelled.
 8. A method as claimed in claim 1 wherein the label is aradioactive label.
 9. A method as claimed in claim 1 wherein the freeligand is a free hormone.
 10. A method as claimed in claim 9 wherein thefree ligand is free thyroid hormone.
 11. A method as claimed in claim 1whereinP₁, P₂, . . . , P_(n) are the endogenous binding agents in thebiological fluid samples, [P₁ ], [P₂ ], . . . [P_(n) ] are theconcentrations of the endogenous binding agents in the biologicalsamples, K_(P), K_(P2) . . . K_(Pn) are the equilibrium constantsbetween the free ligand analogue and the ligand analogue bound to theendogenous binding agents in the biological fluid samples, P_(X) is theexogenous binding agent X, [P_(X) ] is the concentration of the bindingagent X in the biological fluid samples,K_(PX) is the equilibriumconstant between the free ligand analogue and the ligand analogue boundto the binding agent X in the biological fluid samples, and K_(PX)[P_(X) ] is at least 10 and S is not more than 500, where S is 1+ΣK_(P)[P]+K_(PX) [px], ΣK_(P) [P] is K_(P1) [P₁ ]+K_(P2) [P₂ ]+. . . =K_(Pn)[P_(n) ], and is zero or substantially constant in all biological fluidsamples to be assayed.
 12. The method of claim 11 wherein S is 50-500.13. A method as claimed in claim 1 whereinP₁, P₂, . . . , P_(n) are theendogenous binding agents in the biological fluid samples, [P₁ ], [P₂ ],. . . [P_(n) ] are the concentrations of the binding agents in each ofthe biological fluid samples, K_(P1), K_(P2) . . . K_(Pn) are theequilibrium constants between the free ligand analogue and the ligandanalogue bound to the endogenous binding agents in the biological fluidsamples, P_(x) is the exogenous binding agent X, [P_(x) ] is theconcentration of the binding agent X in the biological fluid samples,K_(Px) is the equilibrium constant between the free ligand analogue andthe ligand analogue bound to the binding agent X in the biological fluidsamples, and K_(Px) [P_(x) ] is at least ^(s) /2, where S is 1+ΣK_(P)[P]+K_(PX) [px], ΣK_(P) [P] is K_(P1) [P₁ ]+K_(P2) [P₂ ]+ . . . =K_(Pn)[P_(n) ], and varies significantly from sample to sample of thebiological fluid samples to be assayed, K_(px) [px] being comparablewith or greater than that variation.